Despite slow catalysis and confused substrate specificity, all ribulose bisphosphate carboxylases may be nearly perfectly optimized

Abstract

The cornerstone of autotrophy, the CO(2)-fixing enzyme, d-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco), is hamstrung by slow catalysis and confusion between CO(2) and O(2) as substrates, an "abominably perplexing" puzzle, in Darwin's parlance. Here we argue that these characteristics stem from difficulty in binding the featureless CO(2) molecule, which forces specificity for the gaseous substrate to be determined largely or completely in the transition state. We hypothesize that natural selection for greater CO(2)/O(2) specificity, in response to reducing atmospheric CO(2):O(2) ratios, has resulted in a transition state for CO(2) addition in which the CO(2) moiety closely resembles a carboxylate group. This maximizes the structural difference between the transition states for carboxylation and the competing oxygenation, allowing better differentiation between them. However, increasing structural similarity between the carboxylation transition state and its carboxyketone product exposes the carboxyketone to the strong binding required to stabilize the transition state and causes the carboxyketone intermediate to bind so tightly that its cleavage to products is slowed. We assert that all Rubiscos may be nearly perfectly adapted to the differing CO(2), O(2), and thermal conditions in their subcellular environments, optimizing this compromise between CO(2)/O(2) specificity and the maximum rate of catalytic turnover. Our hypothesis explains the feeble rate enhancement displayed by Rubisco in processing the exogenously supplied carboxyketone intermediate, compared with its nonenzymatic hydrolysis, and the positive correlation between CO(2)/O(2) specificity and (12)C/(13)C fractionation. It further predicts that, because a more product-like transition state is more ordered (decreased entropy), the effectiveness of this strategy will deteriorate with increasing temperature.

Fig. 1.

The formal mechanism of the Rubisco-catalyzed addition of CO₂ and O₂ to enolized RuBP (2, 3, 16, 17). The subscripts of the rate constants are those used previously to describe the mechanism (36). The additions of the gaseous substrates are effectively irreversible (i.e., k₇ and k₄ ∼ 0) (19) and, because enolization is readily reversible (6, 37) and [3-²H]RuBP reduces k_cat^c much less than expected for an intrinsic primary ²H effect (except when enolization becomes rate-limiting at unphysiologically low pH) (38, 39), enolization must be much faster than the maximum catalytic rate (i.e., k₈ and k₅ ≪ k₉). Thus the expressions (36) for the apparent Michaelis constants for CO₂ (K_c) and O₂ (K_o), the maximum carboxylation (k_cat^c) and oxygenation (k_cat^o) rates, the ratios between them (k_cat^c/K_c and k_cat^c/K_o), and the specificity for CO₂ relative to that for O₂ (S_c/o) (40) may be simplified as shown. C₆-int., 2′-carboxy-3-keto-d-arabinitol-1,5-bisphosphate; C₅-int., 2′-peroxy-3-keto-d-arabinitol-1,5-bisphosphate; PGA, 3-phosphoglycerate; PGY, 2-phosphoglycolate.

Fig. 2.

Energetic profile of part of the Rubisco-catalyzed carboxylation reaction, from the carboxylation of the enediolate of RuBP through to the cleavage of the hydrated carboxyketone. Shown are the hypothesized naturally selected shifts (arrows) from a less CO₂-specific Rubisco with a more reactant-like transition state for CO₂ addition (dashed energies) toward a more product-like transition state allowing greater CO₂ specificity (solid energies). The more product-like the transition state for carboxylation [1] is, the more closely it resembles the carboxyketone intermediate [I]. We cannot predict how this will affect the energy level of the whole transition-state-1/active-site complex, so we show a blurred band for the energy at this stage, extending both above and below that of the same transition state in the more reactant-like scenario. Because of their greater resemblance to [1], the carboxyketone intermediate [I] and its gem-diol hydrate [II] are bound more tightly in the more product-like scenario, lowering their energy (see text). As a consequence, the activation energy for cleavage increases, inducing k₈ to decrease. Note that carboxylation and hydration are assumed to be separate steps in this scheme. If they are not, the energetic profiles of CO₂ addition and hydration fuse to one, the hydrated intermediate still having a lowered energy level.

Fig. 3.

Natural variation in the kinetic properties of Rubisco in vitro at 25°C. See Table 1 for a listing of the data and their sources. The organisms from which the Rubiscos were isolated were: Ag, Atriplex glabriuscula (C₃ dicot); Ah, Amaranthus hybridus (C₄ dicot); Av, Anabaena variabilis (cyanobacterium); Cr, Chlamydomonas reinhardtii (green alga); Cv, Chromatium vinosum (bacterium); Eg, Euglena gracilis (green alga); Gm, Griffithsia monilis (red alga); Gs, Galdieria sulfuraria (red alga); Nt, Nicotiana tabacum (C₃ dicot); Os, Oryza sativa (C₃ monocot); Pt, Phaeodactylum tricornutum (diatom); Rr, Rhodospirillum rubrum (bacterium); Rs, the bacterial symbiont of the tubeworm Riftia pachyptila; Sb, Sorghum bicolor (C₄ monocot); So, Spinacia oleracea (C₃ dicot); S6301, Synechococcus PCC 6301 (cyanobacterium); S7002, Synechococcus PCC 7002 (cyanobacterium); Ta, Triticum aestivum (C₃ monocot); and Zm, Zea mays (C₄ monocot). Dashed and dotted lines are linear regressions through all of the data except for the cyanobacterial outliers (triangles) in A and B, which were excluded.

Fig. 4.

The higher the CO₂/O₂ specificity, the more it is eroded by increasing temperature. (A) Arrhenius-style plots of the specificities of the Rubiscos from spinach (•) (32) and the red alga, Galdieria partita (■) (33). (B) Positive correlation between (ΔH_o^‡−ΔH_c^‡) and (ΔS_o^‡−ΔS_c^‡) (Eq. 1) for these Rubiscos plus those from 15 C₃ higher plants (Δ) (34). To remove variation due to the differing effects of temperature on the solubilities of CO₂ and O₂, data for S_c/o at various temperatures were expressed in terms of the gas-phase partial pressures of CO₂ and O₂ (S_c/o^gas) by dividing the reported values (which were expressed in terms of aqueous-solution concentrations) by the ratio between the aqueous solubilities of O₂ and CO₂ appropriate to the measurement temperature (41). The transformed data for each Rubisco were then fitted to Eq. 1 to obtain the estimates of (ΔH_o^‡−ΔH_c^‡) and (ΔS_o^‡−ΔS_c^‡) and their standard errors. For example, (ΔH_o^‡−ΔH_c^‡) and (ΔS_o^‡−ΔS_c^‡) were estimated to be 28.5 ± 0.6 kJ mol⁻¹ and 31.1 ± 2.0 J mol⁻¹°K⁻¹, respectively, for spinach Rubisco; for G. partita Rubisco, the estimates were 51.8 ± 5.3 kJ mol⁻¹ and 102 ± 17 J mol⁻¹°K⁻¹. The lines shown are linear regressions through all of the data.